We have developed a procedure for isolating DNA-dependent RNA polymerase from rooster liver nuclei that involves treatment of nuclei with pancreatic DNAse I to partially solubilize chromatin. The enzyme thus obtained is completely dependent on the addition of exogenous DNA for activity. We have developed a technique for identifying RNA polymerase II in nuclear lysates by binding 3H alpha-amanitin to the polymerase and subsequently subjecting the proteins to electrophorese in polyacrylimide gels under non-denaturing conditions. By this method we have identified two alpha-amanitin binding proteins, tentatively identified as two forms of RNA polymerase II. Unpurified preparations of RNA polymerase II prepared in this manner had the ability to transcribe SV40 DNA asymmetrically in the region of the origin of replication.